Method for tumor treatment using infusion of xenogeneic cells to induce hyperacute rejection and innocent bystander effect

ABSTRACT

A method for treating tumors. Through infusion or xenotransplantation of xenogeneic cells, such as infusion of murine cells into the peritoneal cavity of humans, a hyperacute rejection response to the cells is induced. This in turn creates a bystander effect to the tumor. This effect creates tumor regression. This treatment can be used alone or in conjunction with gene therapy or chemotherapy treatments.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119 of U.S.Provisional Patent Application No. 60/138,038 filed Jun. 8, 1999, andfrom U.S. application Ser. No. 09/589,255 filed Jun. 1, 2000, whichapplications are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Ovarian carcinoma is the most common cause of death from a gynecologicalmalignancy in the United States. Over ⅔ of the patients have advancedstage disease at presentation for which systemic chemotherapy isindicated after surgical debulking. Standard therapy consists ofcisplatin or carboplatin with paclitaxel, and excellent response ratesare observed; however, recurrence is common, and the majority ofpatients still die of disease progression. Ovarian cancer has a fairlyunique natural history in humans. Even patients with advanced stages ofthe disease often have their disease confined to their abdomen forextended periods of time. The disease often stays localized to theabdomen and presents great difficulty for the patient by obstruction ofthe intestines or ureters. This natural history makes the possibility oflocally-targeted therapy realistic. As a result, intraperitonealtherapies have been developed for the local administration ofchemotherapeutic agents into the peritoneal cavity.

Gene therapy is among the experimental strategies for patients withcancer who have failed standard therapy. Despite more than 100 genetherapy trials, evidence of success is very limited. Roth, J. A.,Cristiano, R. J., Roth, J. A. & Cristiano, R. J., Gene therapy forcancer: what have we done and where are we going? J. Natl. Cancer Inst.89(1), 21-39 (1997). One strategy which has been explored for treatingcancer is the artificial creation of differences between normal andneoplastic cells through prophylactic use of gene insertion techniques.In other words, manufacturing biochemical differences which can beexploited to systematically and specifically target neoplastic cells fordestruction. Gene insertion protocols are used to artificiallymanufacture biochemical differences in target tumor cells which are thenexploited to selectively kill these cells. One system which has receivedmuch attention to date is the Herpes Simplex Virus Ganciclovir System.

Transformation of tumor cells with the gene encoding Herpes SimplexVirus thymidine kinase (HSVtk) and subsequent treatment with anti-viralagents such as ganciclovir (GCV) has been previously accomplished andhas proven to be operable in vivo in animals and humans. See GeneTherapy for the Treatment of Recurrent Pediatric Malignant AstrocytomasWith In Vivo Tumor Transduction With Herpes Simplex Thymidine KinaseGene/Ganciclovir System, Raffel, C. et al., Human Gene Therapy 5(7), p.863-90, July 1994. The HSVtk gene is a negative selectable marker or“suicide” gene of which most investigators in the field are well awareand versed in how the system is supposed to function. HSVtk sensitizestransduced tumor cells to GCV. Moolten, F. L., Tumor chemosensitivityconferred by inserted herpes thymidine kinase genes: Paradigm for aprospective cancer control strategy Cancer Res. 46, 5276-5281 (1986);Moolten, F. L. & Wells, J. M., Curability of tumors bearing herpesthymidine kinase genes transferred by retroviral vectors J. Nat. CancerInst. 82, 297-300 (1990); Moolten, F. L., Wells, J. M., Heyman, R. A. &Evans, R. M., Lymphoma regression induced by ganciclovir in mice bearinga herpes thymidine kinase transgene Human Gene Ther. 1, 125-134 (1990);Plautz, G., Nabel, E. G. & Nabel, G. J., Selective elimination ofrecombinant genes in vivo with a suicide retroviral vector New Biologist3(7), 709-715 (1991). GCV is phosphorylated by HSVtk resulting in amonophosphate that cellular kinases convert to GCV-triphosphate whichinhibits DNA replication and causes cell death. An interesting in vitroand in vivo observation with HSVtk is that only a portion of tumor cellsneed to be transduced with this gene to induce complete tumordestruction. This metabolic cooperation is a form of “bystander effect”and is due in large part to the transfer of phosphorylated GCV betweencells through gap junctions. Moolten, F. L., Tumor chemosensitivityconferred by inserted herpes thymidine kinase genes: Paradigm for aprospective cancer control strategy Cancer Res. 46, 5276-5281 (1986);Culver, K. W., Ram, Z., Walbridge, S., Ishii, H., Oldfield, E. H., andBlaese, R. M. In vivo gene transfer with retroviral vector-producercells for treatment of experimental brain tumors Science 256, 1550-1552(1992); Nielsen, C. S., Moorman, D. W., Levy, J. P. & Link, C. J., Jr.,Herpes simplex thymidine kinase gene transfer is required for completeregression of murine colon adenocarcinoma Am. Surg. 63(7), 617-620(1997); Bi, W. L., Parysek, L. M., Warnick, R. & Stambrook, P. J., Invitro evidence that metabolic cooperation is responsible for thebystander effect observed with HSVtk retroviral gene therapy Hum. GeneTher. 4, 725-731 (1993); Freeman, S. M., Abboud, C. N., Whartenby, K.A., Packman, C. H., Koeplin, D. S., Moolten, F. L., and Abraham, G. N.The bystander effect: tumor regression when a fraction of the tumor massis genetically modified Cancer Res. 53, 5274-5283 (1993); Ishii-Morita,H., Agbaria, R., Mullen, C. A., Hirano, H., Koeplin, D. A., Ram, Z.,Oldfield, E. H., Johns, D. G., and Blaese, R. M. Mechanism of ‘bystandereffect’ killing in the herpes simplex thymidine kinase gene therapymodel of cancer treatment Gene Ther. 4(3), 244-51 (1997); Link, C. J.,Jr., Kolb, E. M. & Muldoon, R. R., Preliminary in vitro efficacy andtoxicities studies of the herpes simplex thymidine kinase gene systemfor the treatment of breast cancer Hybridoma 14(2), 143-7 (1995);Samejima, Y. & Meruelo, D., ‘Bystander killing’ induces apoptosis and isinhibited by forskolin Gene Therapy 2, 50-58 (1995); Vrionis, F. D., Wu,J. K, Qi, P., Waltzman, M., Cherington, V., and Spray, D. C. Thebystander effect exerted by tumor cells expressing the herpes simplexvirus thymidine kinase (HSVtk) gene is dependent on connexin expressionand cell communication via gap junctions Gene Ther. 4(6), 577-85 (1997).The implantation of vector producer cells (VPC) to deliver genes, suchas the HSVtk gene, into tumor cells was first demonstrated in a braintumor model by engrafting lacZ VPC into rodents with C6 glioma tumors.Short, M. P., Choi, B. C., Lee, J. K., Malick, A., Breakefield, X. O.,and Martuza, R. L. Gene delivery to glioma cells in rat brain bygrafting of a retrovirus packaging cell line J. Neurosci. Res. 27,427-439 (1990). In these reported animal tumor models, all of therodents are αgal+. Since these were αgal+ models, hyperacute rejectionis not contributing to the observed responses with the HSVtk system. Ahuman, however, is different.

Three prior human trials of murine HSVtk VPC have been reported; two fortreatment of brain tumors and one for melanoma. Results from the firstclinical trial conducted at the NCI using murine VPC xenografts wererecently reported in Nature Medicine. Ram, Z., Culver, K. W., Oshiro, E.M., Viola, J., DeVroom, H. L., Otto, E., Long, Z., Chiang, Y.,McGarrity, G. J., Muul, L. M., Katz, D., Blaese, R. M., and Oldfield, E.H. Therapy of malignant brain tumors by intratumoral implantation ofretroviral vector-producing cells Nature Med. 3, 1354-1361 (1997). Thistrial used multiple stereotaxic injections to introduce murine HSVtk VPCinto the enhancing portion of brain tumors. Anti-tumor activity wasobserved in selected local tumor deposits in 5 of 19 tumors injected in13 patients. Ram, Z. et al., Nature Med. 3, 1354-1361 (1997). It isimportant to note that very limited (minimal) gene transfer wasobserved. The stereotaxic injections required multiple needle passagesto try to distribute the VPC throughout the tumors resulting in severehemorrhage that required surgery in 2 patients and MRI-visible bleedingin most of the other tumors. The authors theorized that the observedresponses were secondary to the cell-to-cell transfer of phosphorylatedGCV as “the major mechanism” of the bystander effect and concluded that“non-immune bystander mechanisms were critical.” Ram, Z. et al., NatureMed. 3, 1354-1361 (1997). They further stated that the observedresponses were “probably not a result of immune mechanisms.” In light ofdata presented in this application and data from others demonstratingrapid destruction of murine VPC by human serum, these conclusions areunwarranted. Rollins, S. A., Birks, C. W., Setter, E., Squinto, S. P. &Rother, R. P., Retroviral vector producer cell killing in human serum ismediated by natural antibody and complement: Strategies for evading thehumoral immune response Human Gene Therapy 7, 619-626 (1996); Link, C.J., Levy, J. P., Seregina, T., Atchinson, R. & Moorman, D., in CancerGene Therapy (eds Mazarakis, H. & Swart, S. J.) 135-152 (IBC LibrarySeries, London, United Kingdom, 1997). We suggest that the contrastenhancement reported along the needle tracts of VPC injections, as wellas the transient volume increase of the tumors immediately afterinjection, was more likely to be the result of antibodies (Ab) andcomplement-mediated hyperacute rejection against the murine cells. Ofnote, the authors did report an increase in VPC binding Ab that peaked21-142 days after VPC injection; these Ab may represent anti-αgal Ab.Ram, Z. et al., Nature Med. 3, 1354-1361 (1997). Other groups have shownthat anti-pig Ab present in human serum are predominantly anti-αgal IgMAb. Vaughan, H. A., McKenzie, I. F. & Sandrin, M. S. Biochemical studiesof pig xenoantigens detected by naturally occurring human antibodies andthe galactose alpha(1,3)galactose reactive lectin Transplantation 59(1),102-9 (1995). The time frame of Ab titer increase is similar to thatreported for diabetic patients transplanted with porcine isletxenografts and with our results with murine VPC intraperitonealinfusions discussed below. Galili, U., Tibell, A., Samuelsson, B.,Rydberg, L. & Groth, C. G. Increased anti-Gal activity in diabeticpatients transplanted with fetal porcine islet cell clustersTransplantation 59 (11), 1549-56 (1995).

A second brain tumor trial was recently reported by Klaztmann andcolleagues. Klatzman, D., Valery, C. A., Bensimon, G., Marro, B., Boyer,O., Mokhtari, K., Diquet, B., Salzman, J.-L., Philippon, J., andGlioblastoma, S.G.o.G.T.f. A phase I/II dose escalation study of Herpessimplex virus type 1 thymidine kinase “suicide” gene therapy forrecurrent glioblastoma Human Gene Ther. 9, 2595-2604 (1998). M11 murineVPC producing HSVtk retroviral vector were injected into the tumormargin after surgical debulking. Seven days later, patients were treatedwith GCV. Twelve patients were treated without side effects that thephysicians attributed to the VPC or GCV. The authors claimed that theobserved responses were secondary to gene therapy. One patient was stillalive without evidence of progression by MRI at 2.8 years after theprocedure. Eleven of twelve patients had died; nine from tumorprogression, one from head trauma, and one from pulmonary embolus. Torelate these observations to the gene therapy, the authors suggestedseveral indirect lines of evidence. First, M11 cells could be recoveredfrom surgical drain fluids 24 hours after cell injections. However, M11VPC were recovered from only 3 out of 6 patients even at this short timepoint. Furthermore, no viable cells were recovered at later time points.Again, neurosurgical procedures with local tumor resections areoperations with active bleeding and oozing into the tumor bed where thecells were injected. In fact, a blood-brain barrier that has been quotedby a number of groups to protect murine VPC (Ram, Z., J. Neurosurg. 79,400-7 (1993)) is mechanically destroyed by the scalpel during majorsurgical debulking. Thus, M11 cells were most likely quickly lysed bythe presence of human serum. The ability to recover a few viable M11cells from the surgical drain 1 day after up to 9.8×10⁶ cells/cm² wereinjected is not compelling evidence that gene transfer is accounting forthese observations. The second indirect evidence proposed was that GCVplasma levels were in a therapeutic range to kill HSVtk expressingcells. It is not understood how the presence of an adequate GCV levelprovides evidence of causation without HSVtk gene transfer data. No genetransfer into glioblastoma cells was reported. The imaging andpathologic data from this trial does suggest that patients who did notdemonstrate MRI progression at 4 months after VPC injection do show someefficacy; however, the relationship between these observed responses andHSVtk and GCV is highly speculative.

One other study was performed on melanoma patients with non-CNSmalignancy. Klatzman, D. et al., Human Gene Ther. 9, 2585-2594 (1998).Eight patients were treated by the direct injection of murine M11packaging cells that produced HSVtk vector. The total cell dose rangedfrom 8×10⁷ to 12.5×10⁸ cells that were directly injected into tumors.Inflammatory reactions were common immediately after these xenogeneicVPC were injected. A rapid increase in tumor size was noted that peaked24 hours later. The investigators attributed these effects to knownpre-existing antibodies against xenogeneic antigens present on murinecells. This suggestion supports our findings. The very limitedanti-tumor effects were some areas of local necrosis noted on biopsysamples. The lack of significant efficacy was attributed to poor genetransfer (<1% or none detected by PCR). Side effects of therapyconsisted chiefly of local inflammatory reactions or fever when multipleinjections were administered. This group suggested that murine VPCsurvival was enhanced by using intravenous immunoglobulin to delayxenogeneic hyperacute rejection. Gautreau, C., Kojima, T., Woimant, G.,Cardoso, J., Devilier, P., and Houssin, D. Use of intravenousimmunoglobulin to delay xenogeneic hyperacute rejection Transplantation9, 903-907 (1995).

Hyperacute rejection of xenografts has been previously explored due tothe great interest in using animals as a source of organs or tissues forhumans. Strong immunological barriers to xenotransplants can destroy atransplanted solid organ within minutes, a process termed hyperacuterejection. This hyperacute rejection model of xenograft survival istypically a vascularized xenograft directly exposed to blood serum.Pruitt, S. K., Kirk, A. D., Bollinger, R. R., Marsh, J., Henry, C.,Collins, B. H., Levin, J. L., Mault, J. R., Heinle, J. S., Ibrahim, S.,Rudolph, A. R., Baldwin, I., William, M., and Sanfilippo, F. The effectof soluble complement receptor type 1 on hyperacute rejection of porcinexenografts Transplantation 57, 363-370 (1994). Research has demonstratedthat hyperacute rejection with porcine xenografts transplanted intobaboons occurs secondary to porcine α(1,3)galactosyltransferase[α(1,3)GT] gene expression and α(1,3)galactosyl epitopes (αgal)presentation. Pruitt, S. K. et al., Transplantation 57, 363-370 (1994);Platt, J. L., Vercellotti, G. M., Dalmasso, A. P., Matas, A. J., Bolman,R. M., Najarian, J. S., and Bach, F. H. transplantation of discordantxenografts: a review of progress Immunol. Today 17, 450-457 (1990). Theenzyme α(1,3)GT is expressed in all mammalian species including Musmusculus, but not in Old World primates, apes, or humans. Galili, U.Shohet, S. B., Kobrin, E., Stults, C. L. & Macher, B. A., Man, apes, andOld World monkeys differ from other mammals in the expression ofalpha-galactosyl epitopes on nucleated cells J. Biol. Chem. 263(33),17755-62 (1988). The α(1,3)GT gene is not active in humans due to thepresence of two base pair frameshift mutations. Larsen, R. D.,Rivera-Marrero, C. A., Ernst, L. K., Cummings, R. D. & Lowe, J. B.,Frameshift and nonsense mutations in a human genomic sequence homologousto a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAcalpha(1,3)-galactosyltransferase cDNA J. Biol. Chem. 265(12), 7055-61(1990). α(1,3)GT catalyzes the transfer of galactose from uridinediphosphate galactose (UDP-Gal) to the N-acetyl-lactosamine acceptors oncarbohydrate side chains in a specific α(1,3)linkage on glycoproteins orglycolipids (Galβ1→4GlcNAc-R).

Galβ1→4GlcNAc-R+UDP-Gal→Galα1→3Galβ1→4GlcNAc-R Anti-αgal Ab present inthe human serum can recognize this epitope. Galili, U. Shohet, S. B.,Kobrin, E., Stults, C. L. & Macher, B. A., J. Biol. Chem. 263, 17755-62(1988). In fact, pre-existing human Ab against αgal represent almost 1%of total human Ab (Galili, U., Evolution and pathophysiology of thehuman natural anti-y-galactosyl IgG (anti-Gal) antibody Springer Semin.Immunopathol. 15, 155-171 (1993)) and are the basis forcomplement-mediated hyperacute rejection. Sandrin, M. S., Vaughan, H.A., Dabkowski, P. L. & McKenzie, I. F. Anti-pig IgM antibodies in humanserum react predominantly with Gal(alpha 1-3)Gal epitopes Proc. Natl.Acad. Sci. U.S.A. 90(23), 11391-5 (1993). Human anti-αgal Ab are thoughtto arise in response to αgal structures on the surface of normal GIflora. The translocation of viable bacteria from the enteric lumen tothe mesenteric lymph nodes is thought to stimulate the host immuneresponse. Neonatal humans and baboons compared to their respectiveadults have very low titers of anti-αgal IgM Ab, the isotype mosteffective in binding complement. Xu, H., Edwards, N. M., Dong, X. &Michler, R. E. Age-related development of human preformed anti-porcineendothelial cell xenoantibody J. Thorac. Cardiovasc. Surg. 15, 1023(1995); Minanov, O. P., Itescu, S., Neethling, F. A., Morgenthau, A. S.,Kwiatkowski, P., and Cooper, D. K. Anti-gal IgG antibodies in sera ofnewborn humans and baboons and its significance pig xenotransplantationTransplantation 63, 182 (1997). Neonatal circulating xenoreactive Ab areof the IgG isotype, presumably attained by placental transfer ofmaternal IgG. Xu, H., Edwards, N. M., Dong, X. & Michler, R. E., J.Thorac. Cardiovasc. Surg. 15, 1023 (1995); Minanov, O. P. et al.,Transplantation 63, 182 (1997); Galili, U., Springer Semin.Immunopathol. 15, 155 (1993). Since newborn gut is sterile, there is atime period in which newborn primates are not exposed to bacterial αgalmoieties and, thus, lack de novo Ab directed against these epitopes.

Murine vector producing cells implanted into humans is another type ofxenograft. It has been demonstrated that murine retroviral VPC and theviral vectors they produce express αgal and, therefore, are lysed by Aband complement within 30 minutes after being exposed to human serum.Link, C. J., Levy, J. P., Seregina, T., Atchinson, R. & Moorman, D.,Cancer Gene Therapy (eds Mazarakis, H. & Swart, S. J.) 135-152 (IBCLibrary Series, London, United Kingdom, 1997); Welsh, R. M., Cooper, N.R., Jensen, F. C. & Oldstone, M. B., Human serum lyses RNA tumor virusesNature 257, 612-614 (1975); Rother, R. P., Fodor, W. L., Springhorn, J.P., Birks, C. W., Setter, E., Sandrin, M. S., Squinto, S. P., andRollins, S. A. A novel mechanism of retrovirus inactivation in humanserum mediated by anti-alpha-galactosyl natural antibody J. Exp. Med.182(5), 1345-55 (1995). The effect of this serum inactivation on VPC andretroviruses is due to αgal expression on the cells. Link, C. J., Levy,J. P., Seregina, T., Atchinson, R. & Moorman, D., Cancer Gene Therapy(eds Mazarakis, H. & Swart, S. J.) 135-152 (IBC Library Series, London,United Kingdom, 1997); Rother, R. P. et al., J. Exp. Med. 182, 1345-55(1995); Rother, R. P., Squinto, S. P., Mason, J. M. & Rollins, S. A.,Protection of retroviral vector particles in human blood throughcomplement inhibition Hum. Gene Ther. 6(4), 429-35 (1995).

Viral vectors can efficiently transduce human tumor cells withanti-tumor therapeutic genes. As part of a study of blocking retroviraldestruction by using human packaging cells, the transfer of the porcineα(1,3)GT to human fibroblasts was shown to result in sensitivity to Aband complement destruction. Collins, M. K., Takeuchi, Y., Cosset, F. L.,Tailor, C. & Weiss, R. A. Development of recombinant retrovirusessuitable for in vivo gene delivery Cold Spring Harbor Meeting: GeneTherapy September 1994, 97 (1994). We have found that the retroviraltransduction of human tumor cells with the α(1,3)GT gene resulted in itsexpression. These human cells displayed αgal and became sensitive tohuman serum. In this project, murine VPC are employed to deliverretroviral vector to intraperitoneal ovarian tumors. We are interestedin the biology of glycosylation and the immunologic effect of αgalepitopes. Therefore, the project goal is to further understandmechanisms of hyperacute rejection. Hyperacute rejection may cause astrong intraperitoneal inflammatory response, that through an innocentbystander mechanism, destroys ovarian cancer cells. The process of localtumor destruction might result in the disruption of tumor anergy. Thishas been a common strategy in gene modification trials. Previousattempts of immunotherapy have mainly employed single cytokine molecules(e.g., IL-2, GM-CSF) (Golumbek, P. T., Lazenby, A. J., Levitskky, H. I.,Jaffee, L. M., Karasuyama, H., Baker, M., and Pardoll, D. M. Treatmentof established renal cancer by tumor cells engineered to secreteinterleukin-4 Science 254, 713-716 (1991); Dranoff, G., Jaffee, E.,Lazenby, A., Golumbek, P., Levitsky, H., Brose, K., Jackson, V., Hamada,H., Pardoll, D., and Mulligan, R. Vaccination with irradiated tumorcells engineered to secrete murine GM-CSF stimulates potent, specific,and long lasting anti-tumor immunity Proc. Natl. Acad. Sci. (USA) 90,3539-3543 (1993); Fearon, E. R., Pardoll, D. M., Itaya, T., Golumbek,P., Levitsky, H. I., Simons, J. W., Karasuyama, H., Vogelstein, B., andFrost, P. Interleukin-2 production by tumor cells bypasses T helperfunction in the generation of an antitumor response Cell 60, 397-403(1990)) or single rejection antigens (e.g., HLA B7, melanoma specifictumor antigen). Nabel, G. J., Nabel, E., Yang, Z., Fox, B. A., Plautz,G. E., Gao, X., Huang, L., Shu, S., Gordon, D., and Chang, A. E. Directgene transfer with DNA-liposome complexes in melanoma: Expression,biologic activity, and lack of toxicity in humans Proc. Natl. Acad. Sci.(USA) 90, 11307-11311 (1993); Reeves, M. E., Royal, R. E., Lam, J. S.,Rosenberg, S. A. & Hwu, P. Retroviral transduction of human dendriticcells with a tumor-associated antigen gene Cancer Res. 56(24), 5672-7(1996).

Since anti-tumor gene therapy requires highly efficient gene transferand expression of therapeutic genes (Roth, J. A., Cristiano, R. J.,Roth, J. A. & Cristiano, R. J. Gene therapy for cancer: what we havedone and where are we going? J. Natl. Cancer Inst. 89(1), 21-39 (1997))and the Goldie-Coldman hypothesis predicts that spontaneous mutations incancer cells provide resistance to chemotherapy and that therapeuticfailures are directly related to tumor burden (Goldie, J. H. & Coldman,A. J. A mathematic model for relating the drug sensitivity of tumors totheir spontaneous mutation rate Cancer Treat. Rep. 63(11-12), 1727-33(1979)), multiple, independent therapeutic targets need to be attackedfor success. The predicted tumor resistance will likely extend to genetherapy as well; for example, our group has demonstrated that resistanceto HSVtk gene and GCV killing is common in tumor cells. Seregina, T.,Levy, J. & Link, C. in Fourth International Conference on the GeneTherapy of Cancer Vol. 2 (eds Sobol, R. E. & Royston, I.) A332 (Appleton& Lange, San Diego, Calif., 1995). In the future, gene delivery methodsthat transfer multiple therapeutic genes in concert or genes withmultiple mechanisms of action will dominate approaches for cancertreatment.

All references cited throughout this application are hereby incorporatedby reference.

Based on the foregoing, it is desirable that a less complicated systemwithout use of vectors be available. Using VPC could be eliminated usingthe present invention, thus, taking away the additional risk to thepatient of using viral vectors. The present invention is able to createanti-tumor responses without the need for gene transfection and additionof a prodrug.

SUMMARY OF THE INVENTION

An object of the invention is a method of tumor treatment.

Another object of the invention is a method for treating cancer byactivation of hyperacute rejection in or near the tumor(s).

A further object of the invention is a method for inducing an immunereaction to attack tumor cells.

These and other objects, features, and advantages will become apparentafter review of the following description and claims of the inventionwhich follow.

The present invention induces hyperacute rejection in and/or in thevicinity of a tumor by xenotransplantation/infusion of xenogeneic cells.The xenogeneic cells activate the hyperacute rejection response tothemselves and induce an immune reaction in which tumor cells aredestroyed. One mechanism of tumor destruction may be by an innocentbystander effect.

It may be possible to add to this effect by infusing cells whichtransfect the tumor cells with a gene that the immune system willrespond to, for example, one which creates α(1,3)galactosyl epitopes ina human system (see, e.g., U.S. Pat. No. 5,869,035, hereby incorporatedby reference).

Additionally, subsequent treatment with a prodrug which is activated bythe gene which was introduced may be added to the therapy. For example,if the HSVtk gene is introduced, ganciclovir may be used (see, e.g.,U.S. Pat. No. 5,631,236, hereby incorporated by reference).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows gels of PCR analysis of patient 4 who received 680 millionmurine LTKOSN.1 vector producer cells (VPC) into the peritoneal cavity.TOP: PCR amplification for the HSVtk gene. Positive controls: LTKOSNplasmid only (10 fg), A375 human melanoma cells mixed with 0.1%, 0.01%,0% A375 cells transduced with LTKOSN vector; intraperitoneal tumorbiopsy from day −1 (before VPC infusion) and 14 days after infusion(before GCV); peritoneal washings obtained at day −1 before and days 3to 35 after VPC infusion. HSVtk gene sequence is detected in the day 14tumor biopsy and on days 3 and 7 after VPC infusion. BOTTOM: PCRamplification for the viral env gene. Positive controls: pPAM plasmidcontaining the viral env gene (10 fg), 0.1% or 0.01% LTKOSN.1 VPC;intraperitoneal tumor biopsy from day −1 and 14; peritoneal washings atday −1, 3, 7, 14 and 35 after VPC infusion. The env gene sequence isdetected in the day 3 and 7 peritoneal washings, but not from the tumorbiopsy. Therefore, VPC are lost from the abdominal cavity after day 7.

FIG. 2 is a graph showing anti-αgal titer after murine LTKOSN.1 VPCinfusion. Data shown for patients 1-9 before (day 0) and on day 14 or 21after infusion.

FIG. 3 is a graph showing killing efficiency of patient serum on murineLTKOSN.1 VPC in vitro.

FIG. 4 is a graph showing killing efficiency of patient peritoneal fluidon murine LTKOSN.1 VPC in vitro.

FIG. 5 is a graph showing the increase in reactivity of patient's serumagainst fetal bovine serum (FSB) following murine VPC LTKOSN.1 infusion(fold).

FIG. 6 is a graph of levels of IL-5 in peritoneal fluid after murineLTKOSN.1 VPC infusion. ELISA assay was performed to measure the levelsof IL-5 from peritoneal washing samples both before and after murine VPCinfusions. Peak levels of IL-5 were found 7 to 14 days after VPCinfusion. This IL-5 response overlaps closely the period during whichimmune destruction and elimination of the cells occurs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

It has been found that there is an anti-tumor response in women withrecurrent ovarian cancer treated with LTKOSN.1 murine VPC and GCV due tothe induced immune effect mediated by the activation of hyperacuterejection against non-primate cells that are infused to produce murineretroviral vectors in situ which is prior to any effect by thetransfection of the cells or activation of the GCV.

Murine cells express a surface glycosylation pattern that is not presenton human cells. The murine α(1,3)galactosyltransferase [α(1,3)GT] enzymeadds α(1,3)galactosyl epitopes (αgal) onto glycoproteins andglycolipids. Pre-existing human antibody can bind these epitopes and fixcomplement resulting in the direct lysis of the murine cells. Our datasuggests that an innocent bystander effect then occurs which induces thedestruction of adjacent cancer cells and possibly stimulates anti-tumorimmunity. Though it is believed the bystander effect is responsible fordestruction of the tumor cells, a different or several mechanisms may beresponsible.

The present invention seeks to shift the current understanding ofpublished trials stating that HSVtk and ganciclovir (GCV) gene therapyis efficacious for the treatment of solid tumors in adults. These trialswere designed with the idea that murine VPC xenografts would provide theproduction of retroviral vectors within solid tumors and efficientlytransfer the HSVtk gene. This gene transfer was then expected tosensitize solid tumors to GCV. Furthermore, a bystander effect caused bymetabolic cooperation was to enhance the effect, so that if only a smallportion of the tumor was transduced with HSVtk, then the entire tumorcould be destroyed. In cell culture and animal models of HSVtk and GCVtherapy, 10-20% of the tumor needed to express the HSVtk gene to obtaincomplete response. Moolten, F. L., Cancer Res. 46, 5276-5281 (1986);Culver, K. W. et al., Science 256, 1550-1552 (1992); Link, C. J., Kolb,E. & Muldoon, R., Hybridoma 14, 143-147 (1995). The implantation of VPCis superior to implanting HSVtk pre-transduced cells alone to increasethe delivery of the HSVtk gene. However, in animal models, this approachis often not successful even when 50% of the cells are pretransducedwith HSVtk for a variety of human tumor xenografts (Link, unpublishedobservations). In a preclinical model of intraperitoneal colonadenocarcinoma treated with LTKOSN VPC, a 50% transduction frequency wasrequired to cure 50% of the animals of syngeneic MC38 tumor grafts.Link, C. J., Kolb, E. & Muldoon, R., Hybridoma. 14, 143-147 (1995).Therefore, high level gene transfer is required in these model systems,and it is very unlikely that the conditions for in vivo gene transfer inhumans would be more favorable. Specifically, these trials have madeclaims that are overly simplistic and presume an effect of HSVtk geneand GCV on tumors. These agents have now been expanded into largertrials in the United States and abroad. Further scientific understandingof the basic human immune response to these murine vector producer cellsshould aid in the further development of VPC technology and provideinsight into this potentially important approach to cancer treatment.

A Phase I trial of 10 patients with recurrent ovarian or fallopian tubecancers was conducted using the xenotransplantation of murine retroviralLTKOSN.1 VPC for in vivo transfer of the HSVtk gene. All patients hadfailed prior therapy with paclitaxel and either cisplatin orcarboplatin. LTKOSN.1 VPC were infused into the peritoneal cavity indoses from 1×10⁶ to 1×10⁸ cell/kg. After 4 weeks, patients were treatedwith GCV for 2 weeks. Viable VPC were recovered from peritoneal washeson day +3 and/or day +7 after infusion at the two highest dose levels.The recovered VPC were still able to produce high titer LTKOSNretroviral vector. VPC were detectable by PCR up to day +7, but at day+14 none were detected. Tumor tissue obtained on day +14 prior to firstGCV injection were positive for the HSVtk gene by PCR. Four of tenevaluable patients demonstrated some evidence of anti-tumor response.One patient had a complete resolution of a 2 cm mass on CT scan and a70% reduction of CA125. Of the remaining 3, first patient had a partial,second patient a minor, and the third patient showed a mixed response tothe treatment. The patient with the mixed response demonstratedsignificant resolution of malignant ascites prior to GCV infusion, butdeveloped a malignant pleural effusion. Quantitative PCR analysisdemonstrated less than 1% gene transfer into intraperitoneal tumorbiopsies prior to GCV administration. All patients were treated asoutpatients.

It is surprising that anti-tumor responses were observed although onlylow level gene transfer was noted. These data suggest the process ofhyperacute rejection associated with the administration of murine VPChas an adverse effect on the intraperitoneal tumor.

Since it is believed that the gene transfer was not necessary to theanti-tumor reaction, it is believed that any xenogeneic cells which willactivate the hyperacute rejection will be effective in the invention.One of ordinary skill in the art would be able to determine which cellsother than murine cells which will be safe and effective. There are manycommercial and academic sources of cell lines. Additionally, one ofordinary skill would be able to develop cells lines to use in thepresent invention.

The Examples show that the xenogeneic cells were infused into theperitoneal cavity in the vicinity of the tumor using a catheter. One ofordinary skill would be able to determine other methods of presentingthe xenogeneic cells to the body in order to induce the hyperacuterejection and bystander effect.

The dose levels of xenogeneic cells that have been used are shown in theExamples. One of ordinary skill would be able to determine a dose levelappropriate for the tumor and the patient which is both safe andeffective.

The prior art method of gene expression and activation of an addedprodrug may be effective used concurrently with the treatment of thepresent invention (hyperacute rejection/bystander effect), this genetherapy/prodrug method may be used as an add-on therapy. Additionally,since it appears that the physiology of the tumor changes afterinduction of the hyperacute rejection and the bystander effect,chemotherapy appears to be effective after the treatment of the presentinvention where the chemotherapy was achieving little, if any, resultsprior to this new treatment. One of ordinary skill would be able todetermine additional treatments which will work prior to, in conjunctionwith, or subsequent to the present treatment.

The vector present in the VPC that was used in the present invention wasLTKOSN. LTKOSN is a retroviral vector derived from the Moloney murineleukemia virus (MoMLV). The vector LTKOSN contains a Herpes Simplex typeI thymidine kinase (HSVtk) gene cDNA that is transcribed from the virallong terminal repeat (LTR) and a bacterial neomycin resistance (neo^(r))gene transcribed from an internal SV40 (simian virus 40) early promoter(LTR-HSVtk-SV-neo^(r)-LTR) in the LXSN backbone. This vector has beenmodified for increased safety by alteration of the gag start codon to astop codon, and by elimination of viral sequences to minimize thepotential for the development of replication-competent virus fromproducer cells which contain the vector.

The present invention was used to treat ovarian and fallopian cancerwhich are solid tumors. One of ordinary skill would be able to determineother tumors which will respond to the treatment of the presentinvention.

The data from the Phase I trial of the present approach did not supportthe published conclusions of researchers (e.g., Ram, Z. et al., NatureMed. 3 1354-1361 (1997); Klatzman, D. et al., Human Gene Ther. 9,2585-2594 (1998)). The current trial was designed to determine ifantitumor responses observed in earlier trials, often attributed byother investigators to HSVtk enzyme activation of GCV, is correct. Thecurrent paradigm theorized that HSVtk gene activation of GCV leads to abystander effect that accounts for observed antitumor responses. Theresults of this investigation may overturn the current paradigm acceptedby investigators in molecular medicine. This finding leads to betterscientific and clinical investigations if VPC is the delivery vehicle.The current investigation also provides insight into novel antitumormechanism (hyperacute rejection) and might be further developed andexploited to benefit cancer patients.

EXAMPLES Example 1 Destruction of Murine Cells by Human Serum AndPeritoneal Fluid

The initial interest in hyperacute rejection resulted from studies ofdestruction of murine vector producer cells (VPC) by peritoneal fluid.VPC have been proposed for in vivo gene delivery as a potentialimprovement over retroviral particles alone to increase the transductionefficiency in tumor models. Short, M. P., Choi, B. C., Lee, J. K.,Malick, A., Breakefield, X. O., and Martuza, R. L., Gene delivery toglioma cells in rat brain by grafting of a retrovirus packaging cellline J. Neurosci. Res. 27, 427-439 (1990). The human serum mediateddestruction of murine VPC was immediate. The results indicated thatsubstantial VPC killing occurred after their exposure to peritonealfluid. It was demonstrated that heparin, enoxaparin, or sCR1 couldspecifically block lysis of murine cells by human serum. Link, C. J.,Levy, J. P., Seregina, T., Atchinson, R. & Moorman, D., Cancer GeneTherapy (eds Mazarakis, H. & Swart, S. J.) 135-152 (IBC Library Series,London, United Kingdom, 1997). SCR1 is a soluble form of complementreceptor 1 that binds activated complement and prevents the attackcomplex from damaging the cell membrane.

Example 2 Phase I Clinical Trial of LTKOSN.1 VPC for Women withRecurrent Ovarian Cancer

A Phase I clinical trial was completed with 10 patients with recurrentovarian or fallopian tube cancer. This study was conducted using thexenotransplantation of murine retroviral LTKOSN.1 VPC for in vivotransfer of the HSVtk gene. All patients had failed prior therapy withpaclitaxel and either cisplatin or carboplatin. All patients weretreated as outpatients. LTKOSN.1 VPC were infused intraperitoneally withdoses from 1×10⁶ to 1×10⁸ cell/kg. After 2 weeks, patients were treatedfor two weeks with ganciclovir (GCV). The data is detailed in Tables 1and 2. TABLE 1 Clinical Outcome in Ovarian Cancer Patients Treated withLTKOSN.1 VPC. Signs Gene Age/ Dose Dose and transfer Patient Stage Tumorlevel (VPC) symptoms observed Result Comments 1 64 Ovarian 1 6 millionabd. Not Partial Local tumor IIIC pain, tested response necrosis onnausea histopathology 2 47 Ovarian 1 57 million abd. No Mixed ResolvedIIIC pain, response ascites before nausea GCV Rx 3 59 Ovarian 1 56million abd. No Progressive Deceased 15 IIIC pain disease mos. aftertreatment 4 51 Ovarian 2 680 million abd. Yes Progressive Deceased 5IIIC pain, disease mos. after fever, treatment nausea, vomiting 5 62Ovarian 2 700 million abd. Not Progressive Deceased 6 IIIC pain, testeddisease mos. after fever, treatment nausea 6 66 Fallopian 2 840 millionabd. Yes Minimal Deceased 8 IIIC pain, response mos. after fevertreatment 7 73 IV Ovarian 3 7 billion abd. No Progressive Receiving paindisease chemotherapy 8 60 Ovarian 3 6.3 billion abd. No Mass lesion CTscan still IIIC pain (2 cm) without regressed, disease 21 CA125 mos.later; decreased ↑CA125 70% 9 63 Ovarian 3 6.2 billion abd. yesProgressive Deceased 3 IIIC pain, disease mos. after nausea, treatmentvomiting 10 52 Ovarian 3 5.7 billion rash, pending Progressive ReceivingIIIC abd. disease chemotherapy pain

TABLE 2 VPC Survival in Patient's Peritoneal Cavity and In Vivo HSVtkGene Transfer in Patients Treated with Escalated Doses of LTKOSN.1Retroviral VPC. Day 14 (pre- Patient number Cell Day 3 Day 7 GCVtreatment) (VPC dose) source HSVtk env VPC (cfu/ml) HSVtk env VPC(cfu/ml) HSVtk env  1 PW − − − − − − − − (1 × 10⁶ cells/kg) TB NA NA NANA NA NA  2 PW − − − − − − − − (1 × 10⁶ cells/kg) TB NA NA NA NA NA NA 3 PW − − − − − − − − (1 × 10⁶ cells/kg) TB NA NA NA NA NA NA  4 PW + +− + + − − − (10 × 10⁶ cells/kg) TB NA NA NA NA NA NA + −  6 PW + + 1.5 ×10⁶ + + 1.3 × 10⁶ − ND (10 × 10⁶ cells/kg) TB NA NA NA NA NA NA + ND  7PW + + − + + − − − (100 × 10⁶ cells/kg) TB NA NA NA NA NA NA − −  8PW + + − + + − − − (100 × 10⁶ cells/kg) TB NA NA NA NA NA NA − −  9PW + +  >1 × 10⁶ + − − + − (100 × 10⁶ cells/kg) TB NA NA NA NA NA NA + −10 PW + Pending − + Pending − + Pending (100 × 10⁶ cells/kg) TB NA NA NANA NA NA + PendingPW: peritoneal wash;TB: tumor biopsy during laparoscopy;−: negative;+: positive;NA—not applicable;ND—not doneMost patients developed some abdominal pain consistent with peritonealirritation that lasted for up to 7 days, and 3 patients had low gradefever. One patient exhibited grade 2 nausea and vomiting, otherwise,only grade 1 toxicities were observed. Four of ten evaluable patientsdemonstrated some evidence of an antitumor response. One patient had thecomplete resolution of a 2 cm mass on CT scan and a 70% reduction ofCA125. Of the remaining three, one patient had a partial, the secondpatient a minor, and the third patient showed a mixed response to thetreatment. The patient with the mixed response demonstrated significantresolution of malignant ascites prior to GCV infusion, but developed amalignant pleural effusion. Tumor tissues obtained on day +14 prior toGCV were positive for the HSVtk gene by PCR in several patients.Quantitative PCR analysis demonstrated less than 1% gene transfer intointraperitoneal tumor biopsies prior to GCV administration. FIG. 1 showsthe results of PCR analysis of peritoneal washes and biopsies conductedon patient 4. The demonstration of tumor regression with only low levelgene transfer (<1%) suggests that hyperacute rejection of the murinecells might inhibit the cancer by an innocent bystander effect such asthat described for red blood cells (RBC) in penicillin antigen-antibodycomplex-mediated hemolytic anemia. Schwartz, R. S., Silberstein, L. E. &Berkman, E. M. Autoimmune hemolytic anemia. In Hemotology BasicPrinciples and Practice, 2^(nd) ed. (eds Hoffman, R. et al.) 723-724(Churchill Livingstone, N.Y., 1995). The penicillin antigen-antibodycomplexes adhere to the RBC membrane and activate complement against theRBC. The agal and anti-αgal antigen-antibody may adhere to tumor cellsand possibly activate complement and injure the tumor cells. ViableLTKOSN.1 murine VPC were recovered from peritoneal wash samples ofpatients on day +3 and/or day +7 after infusion at the two highest doselevels. The recovered VPC cultures were G418 resistant, and theirsupernatants contained high titers of LTKOSN retroviral vector similarto those produced by LTKOSN.1 VPC used for infusion into patients(1-1.5×10⁵ cfu/ml). VPC were detectable by PCR up to day +7, but at day+14 none were detected (FIG. 1)

Example 3 Safety Results

Replication competent retrovirus (RCR)S⁺L⁻ assays were conducted onperipheral blood lymphocytes (PBL) of patients. Cells were co-culturedwith Mus dunni cells for two weeks (four passages). After two weeks,supernatants were collected and centrifuged. The supernatant was thenplaced on PG-4 cells (40 to 60% confluent) for two hours. Thesupernatant was removed and D10 (DMEM, 10% fetal bovine serum,L-glutamine) added. After four to seven days, PG-4 cells were observedfor development of foci. MMLV 4070A was used as the positive control,and Mus dunni cells alone were used as the negative control. No RCR waspresent in PBL samples obtained up to one year after VPC infusion asdetected in co-cultivation with Mus dunni followed by S+L-assay.

Example 4 Sample Treatment and Genomic DNA Isolation

Genomic DNA was isolated from 3 ml of whole blood using the Genomic DNAPurification Kit (Promega or Sigma). Tumors were digested by mincing thetissue and adding tumor lysis solution (2 mg/ml Collagenase, Type II;0.2 mg/ml DNAaseI; 2.1 μg/ml Ilyaluronidase, Type V). The tumor wasdissociated on a slow rocker at RT for 1 hour, and cells were collectedby filtering through a nylon mesh. The peritoneal wash samples werecentrifuged to collect all the cells, and DNA was isolated using theGenomic DNA Isolation Kit (Sigma). Isolation of genomic DNA from A375LTKOSN.1 cells diluted with A375 NV cells was used to create 10⁻², 10⁻³,10⁻⁴, and 10⁻⁵ cell dilutions for PCR controls.

Example 5 Retroviral Envelope Gene Expression (RCR Detection)

MLVENV-F (5′-ACCTGGAGAGTCACCAACC-3′) SEQ ID NO:1 and MLVENV-R(5′-TACTITGGAGAGGTCGTAGC-3′) SEQ ID NO:2 were designed to amplify a 411bp fragment of the env gene. The PCR reaction was 3 min. at 94° C.,followed by 30 cycles of 94° C. for 20 seconds, 68° C. for 1 minute, and72° C. for 1 minute, with a final extension of 10 minutes at 72° C. Thereaction mix was 500 ng of genomic DNA (sample), 25 pmoles of eachprimer, 1×PCR buffer, 0.2 mM dNTPs, 1.25 MM MgCl₂, and 1.25 U Taq. A375NV cells and a sample containing no genomic DNA were used as negativecontrols. 100 fg of pPAM3 was used as a positive control for eachsample. For the lymphocyte samples, 500 ng of each A375 LTKOSN.1dilution of genomic DNA (1×10⁻⁴ and 5×10⁻⁴) was used as additionalcontrols. PCR product from blood lymphocytes and controls weretransferred to membrane using a slot blot. The env probe was labeledwith (³²P)dCTP by the random priming technique (Boehringer Mannheim).The blots were hybridized overnight at 42° C. in Hybridisol (Oncor) andwashed. No env gene sequence was detected by PCR in PBL samples obtainedup to one year after VPC infusion.

Example 6

PCR was Performed to Detect HSVtk Gene in PBL and Tumor Biopsy Samples

PCR primers (JMTKO1 5′ TAT AGA CGG TCC TCA CGG GAT 3′ SEQ ID NO:3 andJMTKO3 5′ TCA TGC TGC CCA TAA GGT AT 3′) SEQ ID NO:4 were designed toamplify a 388 bp fragment of the TK gene. The reaction mix contained 500ng of genomic DNA sample. A375 NV cells and a sample containing nogenomic DNA was used as negative controls. 100 fg of pLTKOSN.1 was usedas a positive control for each sample. For the lymphocyte samples, 500ng of each A375 LTKOSN.1 dilution of genomic DNA (10⁻², 10⁻³, 10⁻⁴, and10⁻⁵) was used as additional controls. 500 ng of 10⁻³ and 10⁻⁴ dilutionsof A375 LTKOSN.1 genomic DNA were used as controls for the tumor andperitoneal wash cells. PCR product from blood lymphocytes and controlswere transferred to membrane using a slot blot. A TK probe was labeledwith (³²P)dCTP by the random priming technique (Boehringer Mannheim).PCR products from peritoneal wash and tumor samples were run out on 1.5%TBE gels and Southern transferred onto nylon membrane followingmanufacturer's instructions. No HSVtk gene transfer into PBL frompatient blood samples up to 3 months after VPC infusion were detected byPCR in any patient.

Example 7 Tracking of Serum Anti-αgal Ab Titers

Patients' serial blood samples were drawn before VPC infusions and thenweekly thereafter. These samples were evaluated by ELISA assay foranti-αgal Ab titers as previously described (FIG. 2). Galili, U.,Tibell, A., Samuelsson, B., Rydberg, L. & Groth, C. G. Increasedanti-Gal activity in diabetic patients transplanted with fetal porcineislet cell clusters. Transplantation 59, 1549-56 (1995). Plates(96-well) were coated with 1 μg α-gal-BSA per well. Serial dilutions ofpatient serum samples were used as the primary antibody, and horseradishperoxidase conjugates of anti-human IgG served as the secondaryantibody. It was expected that patients who received murine cellxenografts would generate increases in anti-αgal titers. Previously,diabetic patients grafted with fetal porcine islet cells had been notedto develop an increase in anti-αgal titers (8 to 64-fold), while totalimmunoglobulin concentration was unchanged. Galili, U., Tibell, A.,Samuelsson, B., Rydberg, L. & Groth, C. G., Transplantation 59, 1549-56(1995). Thus, the increase in anti-αgal titer was the result of aspecific immune response against αgal epitopes on the xenograft. In thefirst 3 patients treated with 1×10⁶ VPC/kg (range 54-57×10⁶ VPC), noincreases in anti-αgal titers were seen. However, strong and significantresponse to the αgal antigen was observed at the higher doses. Patients4 through 9 treated at 10-100×10⁶ VPC/kg dose had 2-fold to 256-foldincreases in anti-αgal titers by ELISA. No significant difference inconcentration of IgG and IgM was found in peripheral blood serum samplesobtained prior and on day 14 following murine VPC infusion (data notshown). Thus, the Ab response is a specific anti-αgal immune response.This might be another way to enhance the immune effects against thetumor and break tolerance. Results from this Phase I clinical trial ofLTKOSN.1 VPC for women with recurrent ovarian cancer suggest a possiblerole for hyperacute rejection in anti-tumor responses in humans.

Example 8 Killing Effect of Patient Serum on LTKOSN.1 Murine VPC InVitro

Aliquots of 1×10⁶ LTKOSN.1 VPC, with viability not less than 95%, weresuspended in incubation medium containing 90% of patient serum and 10%of DMEM (Gibco). The cell suspension was incubated at 37° C. for onehour, and cell viability was assessed using trypan blue exclusion.Patient serum inactivated for 30 minutes at 56° C. served as a negativecontrol for complement-dependent murine VPC destruction. Human A375melanoma cells (1×10⁶/aliquot) served as a control for species-specificanti-αgal antibody-mediated cytotoxicity of patients' sera. Serumsamples were obtained prior to and 7 and 14 days post murine VPCinfusion. The data demonstrated that serum from 7 out of 9 patientskilled nearly 100% of murine VPC in this assay (FIG. 3). By day 28 afterthe infusion of VPC, all patients' sera killed 100% of murine VPC.Viability of A375 cells was not affected by exposure to human serum.Thus, in all patients, sera alone could destroy murine VPC. Thesefindings also support the contention that bleeding during neurosurgicalprocedures would kill murine VPC rapidly.

Example 9 Killing Effect of Patient Peritoneal Fluid on LTKOSN.1 MurineVPC In Vitro

Peritoneal washes were performed during patient evaluation and on days3, 7, and 14 after VPC infusion. Aliquots of 1×10⁶ LTKOSN.1 VPC, withviability not less than 95%, were suspended in incubation mediumcontaining 90% of patient peritoneal fluid and tested as above. Theresults of this analysis over the first 30 days after murine LTKOSN.1VPC were infused is plotted in FIG. 4. Interestingly, the peritonealfluid from only one patient initially killed more than 50′ of the VPC inthis assay. Thus, the pre-existing serum killing ability in the patients(FIG. 3) did not translate into peritoneal fluid that would kill VPCunder these ex vivo experimental conditions. However, over the 30 daysafter murine VPC infusion, the peritoneal fluid was able to mediate highlevel VPC killing. We have been able to show that the anti-agal titerincreases in the peritoneal cavity after VPC infusion (data not shown).Little detectable IgG was present in the peritoneal fluid in mostpatients before the xenotransplant; however, the total IgG in theperitoneal cavity increased dramatically in four of six patients tested.This suggests that the anti-αgal Ab response to murine cells altered thephysiology of the peritoneal cavity. These findings correlate with thetiming of the observed antitumor responses. For example, patient #8 whomanifested the best and most durable anti-tumor response observed had ahigh induced serum titer of anti-αgal Ab (32-fold increase) and a rapidup-slope in the ability of her peritoneal fluid to kill murine VPC inthe ex vivo assay. These data support a correlation between anti-αgalimmunity and tumor response, but in this Phase I trial GCV wasadministered 14 days after VPC, and this temporal overlap makesconclusions about causation difficult. The proposed trial design ofwaiting 4 weeks after VPC infusion to treat with GCV may allow theseeffects to be separated. Changes in the peritoneal cavity may alter theability of the immune system to detect these tumors.

Example 10 Characterization of LTKOSN.1 VPC Obtained from PeritonealWashes

Peritoneal wash samples were centrifuged at 250×g, and the cell pelletswere resuspended in DMEM supplemented with 10% FBS (Gibco) andaliquoted. One mg/ml of G418 (Gibco) was added to one aliquot, and cellswere incubated at 37° C., 5% CO₂. Cells in samples containing VPCreached confluency in approximately 10 days after cell cultureinitiation. Cells were cultured in medium containing one mg/ml G418 fortwo additional weeks, then the supernatant was used to infect IGROVhuman ovarian carcinoma cells in standard vector titration assay asestablished at our Institute. The data on the recovered VPC is shown inTable 2 and clearly demonstrates that high titer VPC can be recovered upto 7 days after intraperitoneal infusions.

Example 11

Anti-Fetal Bovine Serum Response

The humoral immune response to fetal bovine serum (FBS) was also tested(FIG. 5). ELISA for anti-FBS was performed in Nuclon 96-well plates werecoated with BSA by filling wells with 100 μl of 5% BSA in PBS andincubating at 4° C. overnight. The plate was washed four times with PBS,and 150 μl of blocking buffer (1% dry milk; 0.1% SDS) was added andincubated at room temperature for one hour. Primary antibody (100 μldiluted patient serum (1:50) samples followed by serial dilution) wasadded following 4 washes and incubated for one hour. After four washes,150 μl of blocking buffer was added, and the plate was incubated twohours. The secondary horseradish peroxidase-conjugated antibody [100 μlof antibody; 1/40,000 dilution of goat anti-human (Pierce) for patientsamples; 1/15,000 dilution of goat anti-bovine (Kirkegaard & Perry) forpositive control] was added and incubated for one hour. Following fourwashes, 100 μl of room temperature TMB (Sigma) substrate was added andcolor was allowed to develop for 15 minutes. The reaction was stopped byadding 100 μl of 1 M H₂SO₄. Absorbance values were read at 450 nm on anELISA plate reader. Only 3 patients developed anti-FBS Ab (out of 9tested).

Example 12

Human Tumor Cell Clones Susceptible to Serum Killing Express αgal

To demonstrate the significance of α(1,3)GT, our group has previouslydemonstrated that the retroviral transfer of α(1,3)GT into tumor cellscan induce sensitivity to normal human serum. Human tumors are notsensitive to lysis by autologous human serum. A truncated version of themurine α(1,3)GT enzyme was cloned into the LNCX retroviral vectorbackbone. Larsen, R. D., Rajan, V. P., Ruff, M. M., Kukowska-Latallo,J., Cummings, R. D., and Lowe, J. B. Isolation of a cDNA encoding amurine UDPgalactose:beta-D-galactosyl-1,4-N-acetyl-D-glucosaminidealpha-1,3-galactosyltransferase: expression cloning by gene transferProc. Natl. Acad. Sci. U.S.A. 86(21), 8227-31 (1989); Miller, A. D. &Buttimore, C. Redesign of retrovirus packaging cell lines to avoidrecombination leading to helper virus production Molec. Cell Biol. 6,2895-2902 (1986). This eukaryotic expression vector was transfected intohuman A375 melanoma tumor cells that were then exposed to human serum.Many A375 subclones were obtained, and data for three representativeexamples are presented. Clones A375αG.7 and A375αG.8 showed significantsensitivity to serum killing while clone A375αG.11 did not (Table 3).TABLE 3 Effect of human serum on A385αG cells. Human serum Human HumanHeat αgal serum serum + sCR1 inactivated expression Cell line % viable %viable % viable by FACS A375 98.7 Not done 96.9 − A375αG.7 2.6 92.093.9 + A375αG.8 11.1 91.6 95.5 + A375αG.11 96.2 Not done Not done −Incubation with sCR1 prevented cell killing by serum. The sensitiveclones A375αG.7 and A375αG.8 expressed αgal by FACS analysis, whileclone A375αG.11 did not exhibit the epitope. These data show that thepresentation of αgal alone on human tumor cells induces sensitivity tohuman serum.

Investigators have demonstrated that certain types of lectins bindspecifically to agal on glycoproteins or glycolipids. Wood, C., Kabat,E. A., Murphy, L. A. & Goldstein, I. J. Immunochemical studies on thecombining site of two isolectins A4 and B4 isolated from Bandeireasimpliciflia Arch. Biochem. Biophys. 198, 1-11 (1979). This method wasused to quantify the amount of αgal present on cells using Griffoniasimplicifolia isolectin B4 (Vector Laboratories, Burlingame, Calif.) andanalyzed by COULTER FACS analyzer. The presence of agal epitopescorrelated with sensitivity to human serum for the A375αgal clones. Thenext step in acquiring evidence needed to pursue this strategy was todemonstrate in vivo differences in an animal model. This presented aproblem since murine cells contain an intact a(1,3)GT gene. So, weinjected original A375 cells and A375 cells expressing agal into athymicnude mice after brief (30 min) exposure to human serum ex vivo. Animalswere monitored for tumor growth for up to 28 days. Only animals injectedwith A375αG.7 cells exposed to human serum and irradiated A375 cells(positive control) were tumor free. The α(1,3)GT gene can also sensitizecells to normal human serum when transferred by a Herpes simplex vectorinto ovarian or colon carcinoma cells.

Example 13 Eosinphilia Occurs Within the Peritoneal Cavity AfterXenotransplants in Humans

One striking finding in patents was the development of eosinophilia inthe peripheral blood and the peritoneal cavity during time of xenograftrejection (FIG. 6). The peripheral blood eosinophil count increased onaverage by 87% by day 14 after murine cells administration.Intraperitoneal washing cell counts demonstrated a mean of 15% (5-28%)and 13% (1-33%) eosinophils on days 7 and 14, respectively. Eosinophilsmay be important immune effector cells against xenogeneic cellstransplanted into humans. In addition, the level of interleukin-5 (IL-5)was measured in samples of peritoneal fluid from patients both beforeand after the infusion of xenogeneic cells. These data represent aninteresting basis for hypothesis development concerning changes in theimmune physiology of the peritoneal cavity that may lead to immunemediated destruction of ovarian cancer.

Example 14 Phase II Trial

The Phase II trial will involve 43 adult women with recurrent orrefractory ovarian, fallopian, or peritoneal carcinoma, will beevaluated for the extent and location(s) of their disease before beingentered into the study. Patients will have a CT scan and laparotomy orlaparoscopy with biopsy to confirm the diagnosis. During laparoscopy,eligible patients will have a peritoneal dialysis catheter placed. HSVtkVPC will be infused into the peritoneal space over 15-60 minutes in atotal volume of one to two liters of plasmalyte. Four weeks later, GCVwill be administered over one hour at 5 mg/kg/dose IV b.i.d. for 14days. Patients may receive up to three cycles of therapy if no tumorregression is observed. After the completion of therapy, patients whocontinue to show evidence of response will be followed every threemonths for the first year.

Having described the invention with reference to particularcompositions, theories of effectiveness, and the like, it will beapparent to those of skill in the art that it is not intended that theinvention be limited by such illustrative embodiments or mechanisms, andthat modifications can be made without departing from the scope orspirit of the invention, as defined by the appended claims. It isintended that all such obvious modifications and variations be includedwithin the scope of the present invention as defined in the appendedclaims. The claims are meant to cover the claimed components and stepsin any sequence which is effective to meet the objectives thereintended, unless the context specifically indicates to the contrary.

1. A method for treating a solid tumor in an αGal⁽⁻⁾ mammal, havinganti-αGal antibodies, said method comprising: administering to saidmammal at or near said tumor, an effective amount of cells having anα(1,3) galactosyl epitope to activate a local hyperacute rejection,wherein said cells are other than vector producing cells.
 2. The methodof claim 1, wherein the tumor is in the peritoneal cavity.
 3. The methodof claim 1 where the αGal⁽⁻⁾ mammal is a human.
 4. The method of claim 1where the cells having an aGal epitope are of xenogeneic origin,obtained from an αGal⁽⁺⁾ mammal.
 5. The method of claim 4 where saidmammal is of murine origin.
 6. The method of claim 4 where said cellsare from murine origin.
 7. The method of claim 1 where αGal⁽⁺⁾ cells arehuman cells obtained by introduction of a polynucleotide sequenceexpressing the alpha(1,3) galactosyltransferase gene.
 8. The method ofclaim 7 where the alpha(1,3) galactosyltransferase gene is from murineorigin
 9. A method for treating a solid tumor in a human, the methodcomprising: administering at or near said tumor an effective amount ofcells having an α(1,3) galactosyl epitope, said cells beingcharacterized in that they do not have a functional retroviral gag genewherein said cells activate a hyperacute rejection response therebyinhibiting the growth of a tumor in said human.
 10. The method of claim9, wherein the tumor is in the peritoneal cavity.
 11. The method ofclaim 9 where the cells having aGal epitopes are of xenogeneic origin,obtained from an αGal⁽⁺⁾ mammal.
 12. The method of claim 11 where suchmammal is of murine origin.
 13. The method of claim 11 where such cellsare from murine origin.
 14. The method of claim 9 where αGal⁽⁺⁾ cellsare human cells obtained by introduction of a polynucleotide sequenceexpressing the alpha(1,3) galactosyltransferase gene.
 15. The method ofclaim 14 where the alpha(1,3) galactosyltransferase gene is from murineorigin
 16. A method for inhibiting the growth of a solid tumor in ahuman having anti-αGal antibodies, the method comprising: deliveringinto or near the tumor an effective amount of a xenogeneic cell thatexpresses an α (1,3) galactosyl epitope and that does not produceretroviral proteins thereby causing a local hyperacute rejectionresponse against said xenogeneic cell and a bystander immune reactionagainst the tumor thereby inhibiting the growth of the tumor in thesubject.
 17. A method of inhibiting the growth of a solid tumor in ahuman subject, the method comprising: administering to said subject intoor near the tumor an effective amount of xenogeneic cells containing anα(1,3)galactosyl epitope and which do not produce retroviral virions, inorder to activate a hyperacute rejection response thereby inhibiting thegrowth of said tumor.
 18. A method for activating an immune responseagainst a solid tumor in a human subject, the method comprising:administering to said subject into or near the tumor an effective amountof xenogeneic cells containing an α(1,3)galactosyl epitope and which donot produce retroviral virions, without subsequent administration ofganciclovir, where said xenogeneic cells activate an immune responseagainst said tumor.
 19. A method for inhibiting growth of a solid tumorin an animal with pre-existing anti-alpha Gal antibodies comprising:administering to said animal, into or near the tumor, an effectiveamount of xenogeneic cells containing an α (1,3) galactosyl epitope andwhich do not contain a tumor specific antigen specific for said solidtumor, to generate a localized hyperacute rejection response near tosaid solid tumor to inhibit the growth of said tumor.
 20. A method foreliciting an immune response against tumor specific antigens which arepresent within a solid tumor in an animal with pre-existing anti-alphaGal antibodies comprising: administering to said animal, into or nearthe tumor, an effective amount of xenogeneic cells containing an α (1,3)galactosyl epitope and which do not generate retroviral virions, tocreate a localized hyperacute rejection response near said solid tumorand a concomitant immune response against tumor specific antigens withinsaid solid tumor thereby inhibiting the growth of said tumor
 21. Themethod of claim 20 wherein said xenogeneic cells do not contain a tumorspecific antigen for said solid tumor in said animal.
 22. A method foreliciting an immune response against tumor specific antigens which arepresent within a solid tumor in an animal with pre-existing anti-alphaGal antibodies comprising: administering to said animal an effectiveamount of xenogeneic cells containing an α(1,3) galactosyl epitope andwhich do not contain said tumor specific antigen for said solid tumorand do not generate retroviral particles, to create a localizedhyperacute rejection response near said solid tumor thereby inhibitingthe growth of said tumor
 23. A pharmaceutical composition to inhibit thegrowth of a solid tumor in a human subject, said composition comprisingαGal⁽⁺⁾ cells which do not contain a tumor specific antigen present insaid tumor.
 24. The composition of claim 23 where such cells are αGal⁽⁺⁾cells of murine origin.
 25. The composition of claim 23 where such cellsare human cells obtained by introduction of a polynucleotide sequencedirecting the expression of an alpha(1,3) galactosyltransferase gene.26. The composition of claim 25 where such alpha(1,3)galactosyltransferase gene is of murine origin.
 27. The composition ofclaim 26 wherein said cells are inactivated.
 28. The composition ofclaim 23 wherein said αGal⁽⁺⁾ cells are inactivated by ultraviolet,gamma or X irradiation.
 29. The composition of claim 23 wherein saidcells do not contain a retroviral nucleotide sequence.
 30. Thecomposition of claim 23 wherein said cells do not contain a functionalgag gene.
 31. The method of claim 23 wherein said cells do not produceretroviral proteins.
 32. The method of claim 23 wherein said cells areother than vector producing cells.
 33. A method for eliciting an immuneresponse against tumor specific antigens which are present within asolid tumor in an animal with pre-existing anti-alpha Gal antibodiescomprising: administering to said animal an effective amount ofxenogeneic cells containing an α (1,3) galactosyl epitope and which donot contain predetermined tumor specific antigens for said solid tumorand do not generate retroviral particles, to create a localizedhyperacute rejection response near said solid tumor and an immuneresponse to said tumor